9.1.pdf

Binary fission is the main way bacteria reproduce. Before dividing, the cell grows and increases its parts. DNA replication starts at the origin of replication on the chromosome. DNA is copied in both directions until the end of the chromosome (terminus) is reached. The...

Binary fission is the main way bacteria reproduce. Before dividing, the cell grows and increases its parts. DNA replication starts at the origin of replication on the chromosome. DNA is copied in both directions until the end of the chromosome (terminus) is reached. The cell pinches in the middle, forming two new daughter cells, each with a full copy of DNA and cytoplasm. A protein called FtsZ forms a Z ring that marks where the cell will divide. Other proteins join the Z ring to create a structure called the divisome, which builds a wall (septum) between the two cells. Enzymes help break down part of the cell wall to allow new material to form the division wall. Generation time is the time between the same stages in two successive generations. In humans, this is typically 25 years. For bacteria (prokaryotes), the generation time is called doubling time, which is the time it takes for the population to double through binary fission. Doubling times in bacteria vary greatly: E. coli can double in as little as 20 minutes in ideal lab conditions. The same bacteria can take days to double in harsh environments. Most pathogens grow quickly like E. coli, but there are exceptions: Mycobacterium tuberculosis (causes tuberculosis) has a doubling time of 15-20 hours. Mycobacterium leprae (causes leprosy) has a very slow doubling time of 14 days. The Growth Curve Microorganisms in closed cultures (batch cultures) grow in a predictable pattern called the growth curve. No new nutrients are added, and most waste is not removed in these cultures. Example: a pond where cells grow in a closed environment. Culture density refers to the number of cells per unit volume. Infections might or might not follow this growth pattern, depending on the site and type of infection. When live cells are plotted over time, distinct phases in the growth curve are seen. The Lag Phase: Cells are added to fresh nutrient broth (culture medium).  Cells don't increase in number but grow larger and become active. They prepare for the next phase by synthesizing necessary proteins. Any damaged cells repair themselves during this phase. Duration depends on factors like cell type, medium composition, and original cell number. The Log Phase (Exponential Growth): Cells divide rapidly by binary fission, increasing exponentially. The time it takes for cells to divide is called the "intrinsic growth rate." Growth is exponential, not linear, but plotted on a semi-log graph for clarity. Cells are metabolically active and growing at a constant rate. This phase is ideal for industrial and research uses. Cells are most vulnerable to disinfectants and antibiotics during this phase. Stationary Phase: Growth slows due to waste buildup, depletion of nutrients, and lack of oxygen. The number of new cells equals the number of dying cells, so the population becomes stable. Cells switch to survival mode and become less sensitive to antibiotics. Some bacteria form spores, and secondary compounds like antibiotics are produced. Pathogenic bacteria may produce virulence factors (disease-causing agents) in this phase. The Death Phase: Nutrients are depleted, and toxic waste accumulates, causing cells to die. Death exceeds cell division, leading to a decline in cell numbers. Some cells release nutrients that allow surviving cells to form endospores. "Persister" cells, with low metabolic activity, may survive and cause chronic infections. Sustaining Microbial Growth: In a closed environment, nutrients are not added, and waste is not removed. To keep cells in the log phase (important for industry), a chemostat is used. A chemostat provides a steady nutrient supply and removes waste to maintain optimal growth. Measurement of Bacterial Growth Bacterial count: Estimates the number of bacterial cells in a sample. Importance: ○ Indicates the level of infection in clinical samples. ○ Used for quality control in water, food, medication, and cosmetics to detect contamination.  Methods: ○ Direct methods: Physically count the cells. ○ Indirect methods: Measure cell presence or activity without directly counting. Direct Cell Count Direct cell count: Counting cells in a liquid culture or on a plate. Direct microscopic cell count: ○ Uses a calibrated slide (Petroff-Hausser chamber). ○ Count cells under a microscope. ○ Calculate concentration by counting cells in squares and factoring in sample volume. ○ Advantage: Fast, easy, and cheap. ○ Disadvantage: Not effective for very dilute samples, and can't distinguish between live, dead cells, and debris. Fluorescence Staining Viability stains: ○ Differentiate live and dead cells. ○ Green stain: Shows live cells. ○ Red stain: Shows dead cells. Electronic Cell Counting (Coulter Counter) Measures changes in electrical resistance as cells pass through a small opening. Advantage: Rapid and accurate for certain concentrations. Disadvantage: May count multiple cells as one if the sample is too concentrated and doesn’t differentiate live from dead cells. Summary Direct counts provide total cell estimates, but distinguishing between live and dead cells is important for assessing infections, antimicrobial effectiveness, and contamination in food or water. Plate Count: A method used to count live cells by growing them into visible colonies. ○ It measures colony-forming units (CFU) instead of individual cells because multiple cells can form one colony. ○ Limitations: Some bacteria group together, and some don’t grow on plates, leading to an undercount of the actual number of live cells. ○ It’s useful for estimating live bacteria despite its limitations. Counting Colonies: ○ Ideal colony count per plate: 30-300 colonies for accuracy. ○ Too few colonies (300) make results unreliable.  Methods of Inoculating Plates: ○ Pour plate and spread plate are the two main techniques. ○ Both start with a serial dilution to get colonies within the 30–300 range. Serial Dilution Process: Serial Dilution: Involves step-by-step dilution of a culture to reduce the number of cells. ○ Goal: Achieve a plate with 30–300 colonies for accurate counting. ○ Each step dilutes the sample, usually in multiples of 10. ○ Example: Start with 1 mL of culture in 9 mL of solution = 1:10 dilution. Repeat process for further dilutions (e.g., 1:100, 1:1000, etc.). Calculating CFU: ○ Example: If 50 colonies are counted from a 1:10,000 dilution and only 0.1 mL was used, then: 50 × 10 × 10,000 = 5,000,000 CFU/mL. ○ This means there are 5 million cells per mL in the original culture. Challenges: ○ Colony counts over 300 may be inaccurate due to overlapping cells forming one colony. ○ For very dilute samples (e.g., drinking water), membrane filtration is used to concentrate the sample before counting. Important Points: Plate count measures live bacteria by counting colonies. Ideal colony range for accuracy is 30–300. Serial dilution is key to achieving accurate counts. CFU/mL is calculated based on colony counts and dilution factors. Membrane filtration can be used for very dilute samples. The number of microorganisms in very diluted samples is often too low to detect using standard plate count methods. Microbiologists use the Most Probable Number (MPN) method, which is a statistical way to estimate the number of living microorganisms in a sample. This method is often applied to water and food samples, and it detects growth by changes in the cloudiness or color of the sample due to microbial activity. A common use of the MPN method is to estimate the number of coliform bacteria in pond water. Coliforms are bacteria that ferment lactose and indicate water contamination, especially from fecal matter. In this method, the water sample is diluted in three levels and inoculated into lactose broth tubes with different amounts of the sample (10 mL, 1 mL, and 0.1 mL). The broth contains a pH indicator that changes color when lactose is fermented, signaling bacterial growth.  After incubation, growth is measured by observing color changes: ○ In the 10-mL tubes, all tubes showed growth. ○ In the 1-mL tubes, only 2 out of 5 tubes showed growth. ○ In the 0.1-mL tubes, no growth was observed. The results (5, 2, and 0) are compared with a probability table, which suggests there are 49 bacteria per 100 mL of pond water. Important Points in Bullet Form: Microorganisms in dilute samples may not be detectable with plate count methods. The Most Probable Number (MPN) method estimates the number of viable microorganisms statistically. MPN is used for water and food samples, based on observing changes in turbidity (cloudiness) or color. Example: MPN method estimates coliform bacteria (lactose-fermenting bacteria) in pond water. Coliforms are a sign of water contamination, especially from fecal matter. Procedure: ○ 3 dilutions of the water sample (10 mL, 1 mL, and 0.1 mL). ○ Each sample is placed in lactose broth tubes (5 tubes per dilution). ○ A pH indicator in the broth changes color when lactose is fermented (red to yellow). ○ Growth results: 10-mL sample: all 5 tubes show growth. 1-mL sample: 2 out of 5 tubes show growth. 0.1-mL sample: no tubes show growth. Using a probability table, the result suggests 49 bacteria per 100 mL of pond water. Indirect Cell Counts Turbidity (cloudiness) of a bacterial sample in liquid can indicate cell density. A spectrophotometer measures turbidity by transmitting light through a bacterial suspension. Less light passes through as bacterial numbers increase, resulting in: Lower percent transmission Higher absorbance (optical density) Measuring turbidity is a quick way to estimate cell density. Viable plate counts can correlate turbidity readings with actual cell numbers, generating a calibration curve. A calibration curve helps estimate cell counts for future samples. Another method: Measuring dry weight of cells by filtering, washing, and drying them. Useful for filamentous microorganisms that are hard to count. Indirect ways to measure live cells include: Monitoring ATP formation, protein/nucleic acid biosynthesis, or oxygen consumption. These methods are fast and easy compared to growing cells. Alternative Patterns of Cell Division Binary fission is the most common type of cell division in prokaryotes, but other methods exist. Other division methods include asymmetrical division (like budding) or producing spores in filaments. In cyanobacteria, many nuclei gather in a round cell or filament, leading to many new cells forming at once. These cells split from the parent in a process called fragmentation. Fragmentation is common in Actinomycetes, a type of soil bacteria. The giant bacterium Epulopiscium exhibits a unique form of division, where daughter cells grow inside the parent, which then breaks apart to release the new cells. In budding, a smaller cell (bud) forms from a long extension on the parent and eventually detaches. This is common in yeast, prosthecate bacteria, and cyanobacteria. Actinomyces (soil bacteria) grow in long filaments that divide into long cells with multiple nuclei. Under low nutrient conditions, they form aerial filaments where cells divide to create spores, which form new colonies. Biofilms: Microorganisms mainly grow in biofilms, which are complex ecosystems on various surfaces. Biofilms form on solid surfaces (e.g., rocks, pipelines) and in liquids with minimal nutrients. Examples include floating microbial mats in water and biofilms in the human mouth, which can have hundreds of bacterial species. Biofilms are structured communities that provide advantages to microorganisms. Biofilm Structure: Biofilm structure changes based on environmental conditions. In fast-moving water, biofilms form streamers, with a head attached to a surface and a tail floating in the current. In slow-moving water, biofilms take on a mushroom-like shape. Biofilms have clusters of microorganisms embedded in an extracellular matrix (EPS), which makes up 50%-90% of the biofilm mass. EPS is made mostly of polysaccharides and contains proteins, nucleic acids, and lipids. EPS helps maintain the biofilm’s structure, keeps it hydrated, and protects microorganisms from predators. Biofilm Formation: Free-floating cells (planktonic cells) attach to a surface, becoming sessile (attached). The attachment starts with a reversible binding, but then cells produce EPS, making the attachment permanent. The biofilm develops a matrix and water channels. Structures like fimbriae, pili, and flagella interact with the EPS to form a mature biofilm. At the final stage, some cells revert to a planktonic state, dispersing to colonize new areas. Biofilm Dynamics: Microorganisms in a biofilm collaborate metabolically. Waste from one species serves as a nutrient for another. Aerobic organisms (requiring oxygen) create anaerobic (no oxygen) zones, allowing anaerobic organisms to thrive. Quorum Sensing: Cells in a biofilm communicate through a process called quorum sensing. This involves small molecules called autoinducers that allow microorganisms to sense their population density. When a critical population is reached, genes beneficial to survival are activated. This can trigger the production of factors that help pathogens evade the immune system. Communication Molecules: Gram-negative bacteria use specific molecules (N-acylated homoserine lactones). Gram-positive bacteria use small peptides. Binding of autoinducers to receptors triggers a signaling cascade affecting gene expression. Impact on Human Health: Biofilms can be beneficial (e.g., gut microbiota) or harmful (e.g., dental plaque, infections). Pathogens in biofilms (e.g., Pseudomonas aeruginosa in cystic fibrosis) are more resistant to antibiotics. Resistance mechanisms include: ○ Metabolically inactive cells in deeper biofilm layers. ○ Extracellular polymeric substances (EPS) that block antibiotics. ○ Increased efflux pumps that expel antibiotics from cells. ○ Exchange of antibiotic resistance genes within biofilms. Biofilms in Medical Devices and Equipment:  Biofilms can clog and compromise devices (e.g., CPAP machines, filtration systems on the ISS). Traditional anti-bacterial treatments often fail when biofilms establish. Innovative Solutions: Researchers have developed a lubricant treatment to prevent biofilm adhesion. This approach is effective both on Earth and in space, with potential applications in medical devices and other areas.